An ever-enlarging world population has increased demands on water and food resources worldwide. Indeed, this population increase is directly proportional to the potential for surface and ground water contamination by pathogenic organisms associated with increased waste burdens. In addition, modern high-output production of meat and other foods represents significant sources of food-borne illnesses. To ensure good public health, there is a need for readily available methods to detect and enumerate pathogens in water, feed, and food. Unfortunately, despite years of testing and research, no single procedure is available for the reliable detection of the major waterborne and foodborne pathogens. Indeed, there are no standardized methods for detecting all of the important pathogens associated with food and waterborne disease. The methods that are available are usually time-consuming and expensive.
I. Gastrointestinal Diseases
Despite advances in public health technology, water and food remain important reservoirs of diarrheal and other diseases of humans and other animals. Infectious diarrhea among children (especially those under 5 years of age), the elderly housed in nursing homes, and travellers visiting developing countries represents a significant public health concern. According to one estimate, infectious diarrhea results in the hospitalization of 200,000 children in the United States each year, at an annual cost of one billion dollars (M. Ho et al., "Rotavirus as a cause of diarrheal morbidity and mortality in the United States," J. Infect. Dis., 158:1112-1116, 1988).
A. Water-Borne Disease
Worldwide, waterborne disease is of even greater significance, with over 250 million reported cases of waterborne disease and more than 10 million deaths annually (J. D. Snyder and M. H. Merson, "The magnitude of the global problem of acute diarrheal disease: a review of active surveillance data," Bull. World Health Organ., 60:605-613 [1982]). When other sources of diarrheal disease are taken into consideration the figures are even more staggering, with these diseases claiming the lives of over 5 million children per year in developing countries (T. L. Hale, "Genetic basis of virulence in Shigella species," Microbiol. Rev., 55:206-224 [1991]).
Most of the cases of waterborne diarrheal disease result from the contamination of drinking water supplies with human fecal material. Contamination of ground water in local areas may occur through such mechanisms as seepage of sewage into aquifers and by improperly developed or poorly protected wells. When factors such as recreational exposure to contaminated salt and fresh water are also taken into consideration, diarrheal disease takes on even greater importance.
Various infectious agents are associated with human waterborne diseases, including Campylobacter, E. coli, Leptospira, Pasteurella, Salmonella, Shigella, Vibrio, Yersinia, Proteus, Giardia, Entoamoeba, Cryptosporidium, hepatitis A virus, Norwalk, parvovirus, polio virus, and rotavirus. Worldwide, the most common bacterial diarrheal diseases are associated with waterborne transmission of Shigella, Salmonella, pathogenic E. coli, Campylobacter jejuni, and Vibrio cholerae (Singh and McFeters, "Detection methods for waterborne pathogens," pp. 125-156, in R. Mitchell (ed.), Environmental Microbiology, [Wiley-Liss, New York, 1992]). Table 1 lists important characteristics of diseases associated with a few of the most significant organisms.
TABLE 1 ______________________________________ Waterborne Diarrheal Bacterial Diseases Most Commonly Reported Incubation Organism Disease Period Common Symptoms ______________________________________ Shigella sp. Shigellosis 1-7 days Diarrhea, fever, cramps, tenesmus, dysentery* Salmonella Salmonellosis 6-72 hours Abdominal pain, diarrhea, nausea, vomiting, fever S. typhi Typhoid fever 1-3 days Abdominal pain, fever, chills, diarrhea or constipation, intestinal hemorrhage Pathogenic Diarrhea 12-72 hours Diarrhea, fever, E. coli vomiting* Campylobacter Gastroenteritis 1-7 days Abdominal pain jejuni suggesting acute appendicitis, fever, headache, malaise, diarrhea, vomiting Proteus sp. Scombroid fish Few minutes Headache, dizziness, poisoning to 1 hour vomiting, nausea, peppery taste, burning throat, facial swelling and flushing, stomach pain, itching Yersinia Yersiniosis 24-36 hours Severe abdominal enterocolitica pain, fever, headache Vibrio 12 hours Vomiting, diarrhea, parahaemolyticus abdominal pain, fever Vibrio cholerae Gastroenteritis 1-3 days Vomiting, diarrhea, dehydration ______________________________________ *Additional symptoms and sequelae are discussed below.
Swimming-associated outbreaks caused by Shigella, Giardia, Norwalk-like viruses, and other enteroviruses have been well documented (See e.g., Makintubee et al., "Shigellosis outbreak associated with swimming," Am. J. Public Health 77:166-168 [1987]; F. J. Sorvillo et al., Shigellosis associated with recreational water contact in Los Angeles County," Am. J. Trop. Med. Hyg., 38:613-617[1988]).
The following table lists the majority of waterborne infectious bacteria which are associated with human diarrheal and non-diarrheal disease.
TABLE 2 ______________________________________ Infectious Bacteria Transmitted by Water Commonly Associated Organism Diseases in Humans ______________________________________ Acinetobacter calcoaceticus Nosocomial infections Aeromonas hydrophila Enteritis, wound infections A. sobria A. caviae Campylobacter jejuni Enteritis C. coli Chromobacterium violaceum Enteritis Citrobacter spp. Nosocomial infections Clostridium perfringens, type C Enteritis Enterobacter spp. Nosocomial infections E. coli, various serotypes Enteritis** Flavobacterium meningosepticum Nosocomial infections, meningitis Francisella tularensis Tularemia Fusobacterium necrophorum Liver abscesses Klebsiella spp. Nosocomial infections, pneumonia Leptospira icterohaemorrahagia Leptospirosis and other Leptospira spp. Legionella pneumophiia Legionellosis and other Legionella spp. Morganella morganii Urethritis, nosocomial infections Mycobacterium tuberculosis Tuberculosis M. marinum Granuloma, dermatitis and other Mycobacterium spp. Plesiomonas shigelloides Enteritis Pseudomonas pseudomallei Melioidosis Pseudomonas spp. Dermatitis, ear infections Salmonella enteritidis Enteritis (salmonellosis) S. montevideo B S. typhimurium and other Salmonella serotypes S. paratyphi A and B Paratyphoid fever S. typhi Typhoid fever Serratia marcesens Nosocomial infections Shigella spp. Dysentery Staphylococcus aureus Wounds, food poisoning Vibrio cholerae Cholera V. alginolyticus Enteritis V. fluvialis Wound infections V. mimicus V. parahaemolyticus V. vulnificus Other Vibrio spp. Yersinia enterocolitica Enteritis ______________________________________ *After T. C. Hazen and G. A. Toranzos, "Tropical Source Water," p. 33, in G. A. McFeters, Drinking Water Microbiology [SpringerVerlag, New York, 1990]. **Additional diseases and sequelae are discussed below.
While the presence of pathogens in drinking and recreational waters presents a significant public health concern, recovery of pathogens from environmental samples is generally difficult. In addition to the usually low numbers of organisms present in the water, nutrient limitations and environmental stressors produce unpredictable physiological and morphological changes in these pathogens. This makes their recovery, isolation and identification problematic. Organisms injured due to these environmental stressors often exhibit atypical reactions and require specialized handling for their resuscitation (see e.g., Singh and McFeters, p. 132-133).
Routine or periodic monitoring of water for the presence of pathogens is essential in situations such as wastewater reclamation, during and after waterborne outbreaks, and for water sources with a frequent history of contamination. This is largely due to the observation that most enteric pathogens appear intermittently and in low concentrations in aquatic environments. Thus, potentially pathogenic organisms may be present in a water supply and go undetected, largely due to their low numbers and the limitations of current testing methods, including relatively low sensitivity levels.
Often, organisms are present but are unculturable (Singh and McFeters, at 131-159; see also, J. J. Byrd et al., "Viable but nonculturable bacteria in drinking water," Appl. Environ. Microbiol., 57:875-878 [1991]; C. Desmonts et al., "Fluorescent-antibody method useful for detecting viable but nonculturable Salmonella spp. in chlorinated wastewater," Appl. Environ. Microbiol., 56:1448-1442 [1990]; and J. J. Byrd and R. R. Colwell, "Maintenance of plasmids pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment," Appl. Environ. Microbiol., 56:2104-2107 [1990]). Unless other methods are used for their detection (e.g., immunoassays) these viable, but non-culturable organisms may present an undetected threat to public health. In addition, the methods commonly used to detect these pathogens were initially designed for clinical, rather than environmental samples. This is of significance in view of the different ecological niche occupied by clinical as compared with environmental isolates. Clinical isolates are usually provided needed nutrients by their host animal and are generally protected from harsh environmental conditions such as cold, heat, damaging chemicals and radiation. In contrast, environmental isolates must deal with these environmental conditions and effectively compete with organisms naturally present and adapted to life in the environment.
B. Food-Borne Disease
In addition to water, food represents an important source of pathogens for humans and other animals. Food-borne illnesses represent an important cause of morbidity, mortality and economic loss in the United States, as well as other countries. Recent estimates by the U.S. Department of Agriculture (USDA) indicate that bacteria are responsible for 3.6 to 7.1 million cases of food-borne illness annually, with associated medical costs and productivity losses of $2.9 to 7.1 billion (J. G. Morris, "Watching the birds (and the beef): New approaches to meat and poultry inspection," ASM News 61:56-57 [1995]). Costs to affected individuals include medical bills, time lost from work, pain and inconvenience. Costs to the food industry include possible product recalls, closing and cleaning of food processing establishments, higher premiums for product liability insurance, loss of product reputation and reduced demand. Approximately $300 million/year is spent by the Federal public health sector on microbial food-borne diseases; federal costs average about $200,000 per food-borne illness outbreak (USDA, "Pathogen Reduction; Hazard Analysis and Critical Control Point (HACCP) Systems," Federal Register Part II 60(023):6774 (Friday, Feb. 3, 1995); hereinafter "USDA").
Food handling from harvest or slaughter to consumption provides numerous opportunities for contamination with pathogenic microorganisms. In the early 1890's, recognition of potential safety problems associated with meat led to enactment of inspection laws in Europe and the United States. These laws have undergone periodic revision and federal laws to protect the food supply are currently in force. Indeed, the laws require that inspected meat and poultry products bear an official inspection legend. Presently, more than 7,300 inspectors from the Food Safety and Inspection Service (FSIS) of the USDA enforce the inspection laws in approximately 6,2000 meat and poultry establishments. Inspection activities start prior to slaughter and continue through processing, handling and packaging. Of the 129,831,110 meat-animal carcasses inspected during fiscal year 1993, 384,543 (0.3%) were condemned for disease, contamination or adulteration during the inspection process. Of the 7,085,491,852 poultry carcasses inspected this same year, 63,926,693 (0.9%) were condemned (USDA, at 6780).
Unsanitary practices and working conditions (e.g., in slaughterhouses), as well as improper storage and preparation procedures contribute greatly to the risk of contamination. For example, at least seventy pathogens have been identified among the organisms isolated from animals at slaughterhouses (J. G. Black, Microbiology Principles and Applications, 2d edition, Prentice Hall, New Jersey, [1993] p. 751). In many areas of the United States, poultry and eggs are commonly contaminated with Salmonella. Over twenty bacterial genera have been isolated from dressed poultry, and improperly handled poultry accounts for many cases of foodborne disease. Half of the infections associated with consumption of improperly handled poultry obtained at restaurants are due to Salmonella, while one-fourth are due to Clostridium perfringens, and the remaining one-fourth are due to S. aureus (Black at p. 751-752). This is of particular concern when the large number of birds slaughtered annually is taken into consideration (over 6 billion chickens and turkeys are slaughtered in the U.S. each year (USDA, "Pathogen Reduction; Hazard Analysis and Critical Control Point (HACCP) Systems," Federal Register Part II 60(023):6774 (Friday, Feb. 3, 1995)). In a study conducted in 1990-1992, approximately 25% of raw products, including broiler chickens were contaminated with Salmonella. In a study on ready-to-cook raw beef conducted from January 1987 through March 1990, the prevalence of Salmonella in 25 gram samples was found to be 1.6%, the prevalence of Listeria was 7.1%, and the prevalence of E. coli 0157:H7 was 0.1%. In a 1992 study of heifer and steer carcasses, C. perfringens was recovered from 2.6% of 2,079 carcasses, C. jejuni/coli was recovered from 4% of 2,064 carcasses, S. aureus was recovered from 4.2% of 2,089 carcasses, E. coli 0157:H7 was recovered from 0.2% of 2,081 carcasses, and Salmonella was recovered from 1% of 2,089 carcasses.
It has been estimated that food-borne pathogens account for up to 7 million cases of food-borne illness annually in the U.S., and up to 7,000 deaths. Of these, almost 5 million cases of foodborne illness and more than 4,000 deaths may be associated with meat and poultry products contaminated with pathogens (USDA, at 6781-6782). However, these estimates may be too low. The following table lists selected food-borne pathogens (including one protozoan parasite, Toxoplasma gondii), the number of cases which were reported in 1993, and estimates of the associated costs. The USDA Economic Research Service and the Centers for Disease Control (CDC) estimate that the cost of all of all food-borne illness in the U.S. in 1993 to have been between $5.6 and $9.4 billion. Of these cases, meat and poultry products were associated with approximately $4.5 to $7.5 billion.
TABLE 3 __________________________________________________________________________ Selected Food-Borne Pathogens 1993 Food- Total Cases Total Deaths borne Costs Percent from Pathogen in 1993 in 1993 (bil $) Meat/Poultry __________________________________________________________________________ Campylobacter 2,500,000 200-730 0.6-1.0 75 jejuni or C. coli Clostridium 10,000 100 0.1 50 perfringens E. coli 0157:H7 10,000-20,000 200-500 0.2-0.6 75 Listeria monocytogenes 1,795-1,860 445-510 0.2-0.3 50 Salmonella 800,000-4,000,000 800-4,000 0.6-3.5 50-75 Staphylococcus 8,900,000 7,120 1.2 50 aureus T. gondii 4,111 82 2.7 100 __________________________________________________________________________
As illustrated by the above table, Salmonella was associated with a large percentage of food-borne illness cases and deaths. However, other organisms are also commonly associated with foodborne infection and disease. For example, many of the organisms listed in Table 1 above are associated with foodborne disease. The following Table lists the organisms most commonly isolated from cases of foodborne disease or infection, foods commonly associated with the organisms, and their reservoirs of infection. Additional organisms, such as the Helicobacter species are also associated with gastrointestinal disease (although the mode of acquisition and transmission of this genus is not clear).
TABLE 4 ______________________________________ Foodborne Bacterial Diseases Most Commonly Reported Commonly Associated Reservoir Organism Disease Food(s) of Infection ______________________________________ Shigella sp. Shigellosis Salads, shrimp, Human ice Salmonella sp. Salmonellosis Poultry, eggs, Poultry, cattle, powdered milk, sheep, turtles, chocolate, humans sausage S. typhi Typhoid fever Foods contaminated Humans with feces of carriers Pathogenic Various Soft cheese, Various foods, E. coli ground meat infected humans and other animals Staphylococcus Staphylococcal Cream-filled Nose, skin, aureus food intoxication pastries, ham, and lesions on pork, sausage humans, udders of cattle Clostridium Gastroenteritis Gravies, meat Soil, humans perfringens C. botulinum Botulism Canned foods, Soil fish products, potatoes Bacillus cereus Gastroenteritis Boiled and fried Soil rice, custards, sauces, meatloaf, gravies Streptococcus Streptococcal Raw milk, dairy Nose and pyogenes infections products, salads, throat of uncooked foods humans, infected sores Listeria Listeriosis Raw milk, Intestinal tract monocytogenes cheese, of humans and contaminated other animals, vegetables soil Brucella spp. Brucellosis Raw milk, goat Cattle, pigs, cheese sheep, goats Campylobacter Gastroenteritis Foods of animal Raw milk, jejuni origin poultry, beef, pork Proteus spp. Scombroid fish Tuna, mackerel, Humans, ice poisoning mahi mahi Yersinia Yersiniosis Dairy products, Cattle, ice enterocolitica meats Vibrio Gastroenteritis Marine animals Raw sea fish, parahaemo- shellfish lyticus Vibrio cholerae Gastroenteritis Raw fish and Fish and shellfish grown in shellfish from or washed with contaminated contaminated water water Vibrio Wound Raw seafood, sea Seafood and vulnificus and infections, water seawater other Vibrio septicemia spp. Aeromonas Diarrhea, Dairy products, Meats, spp. dysentery meats, produce, produce, dairy, bacteremia, water, marine marine wound infections, products environment endocarditis, meningitis, pneumonia, osteomyelitis, peritonitis, conjunctivitis, thrombophlebitis, cholecystitis Plesiomonas Gastroenteritis Freshwater, Seafood, shigelloides seafood, water, amphibians, amphibians, reptiles reptiles Helicobacter Gastritis, ? ? pylori ulcers, H. fennelliae bacteremia, H. cinaedi neonatal septicemia and meningitis, proctocolitis, proctitis ______________________________________
While colonization of the gastrointestinal tract by pathogens occurs and may cause disease (e.g., bacillary dysentery due to Shigella, diarrhea due to Salmonella, typhoid, etc.), many of the bacterial diseases associated with the gastrointestinal tract are due to ingestion of pre-formed toxins present in foods (e.g., toxins produced by Staphylococcus aureus, Bacillus cereus, Clostridium botulinum, etc.). In addition, many organisms which have colonized the gastrointestinal tract produce toxins which cause the signs and symptoms of disease (e.g., cholera, antimicrobial-associated pseudomembranous colitis due to Clostridium difficile, etc). Thus, the type of disease experienced by a patient often mandates the type of specimen(s) collected and the timing of such collections. Recently, some serotypes of E. coli, normally a harmless commensal resident in the intestinal tract of mammals have been recognized as important pathogens.
Concern over food safety largely due to recent outbreaks such as the salmonellosis outbreak associated with contaminated ice cream in Minnesota and the E. coli 0157:H7 outbreaks associated with fast food restaurants has lead to recent USDA Food Safety and Inspection Service (FSIS) proposals to implement a hazard analysis and critical control point (HACCP) strategy to assure safety of poultry and other meat (USDA, "Pathogen Reduction; Hazard Analysis and Critical Control Point (HACCP) Systems," Federal Register Part II 60(023):6774 (Friday, Feb. 3, 1995) (See J. L. Fox, "USDA's food-safety push boosts assay makers," Bio/Technol., 13:114-115, [1995]). These proposed regulations represent a major change in the currently used inspection practices. If enacted, these regulations would require food producers to identify particular steps along the production path where problems such as microbial and chemical contamination of food is likely to occur. Once these "critical control points" are identified, this would permit the food producers to monitor their safety efforts and take appropriate measures to prevent or correct problems.
Of concern in development of new tests to monitor food safety is the lack of specificity associated with antibody-antigen recognition systems and the lack of sensitivity of amplification methods (J. L. Fox, at 115). In addition, some of the molecular tests are difficult to use and are not readily adaptable to the environmental setting. Thus, acceptance of such methods by food producers remains questionable. What is needed is a method which can provide rapid, accurate identification of specific pathogens such as E. coli 0157:H7 and Salmonella on the surface of raw food products, at a minimal cost.
C. Clinical Samples Associated With Diagnosis of Gastrointestinal Disease
In addition to food and water, clinical samples are commonly tested for the presence of these organisms. Indeed, the diagnosis of infectious disease has traditionally relied upon microbiological culture methods to identify the causative organism and determine the appropriate antimicrobial treatment. This has remained so despite recent advances in molecular and immunological diagnostics. While the development of rapid and automated methods has served to increase the efficiency of microbiological analysis, traditional quantitative culture methods remain critical for definitive diagnosis of urinary tract and other infections (E. J. Baron & S. Finegold, Diagnostic Microbiology, 8th ed., C. V. Mosby, [1990], p. 253).
With respect to the type of specimen, there are considerations related to the normal flora from which the pathogens must be differentiated. This is particularly true for fecal, rectal, vaginal, buccal and other samples which commonly contain a characteristic background flora. For other samples (e.g., food, milk, water, and environmental), background flora and other considerations must also be taken into account. This is especially important with E. coli, as it is a commensal intestinal organism that is routinely isolated from healthy individuals.
II. Echerichia coli as a Pathogenic Organism
E. coli is the organism most commonly isolated in clinical microbiology laboratories, as it is usually present as normal flora in the intestines of humans and other animals. It is also an important cause of intestinal, as well as extraintestinal infections. For example, in a 1984 survey of nosocomial infections in the United States, E. coli was associated with 30.7% of the urinary tract infections, 11.5% of the surgical wound infections, 6.4% of the lower respiratory tract infections, 10.5% of the primary bacteremia cases, 7.0% of the cutaneous infections, and 7.4% of the other infections (J. J. Farmer and M. T. Kelly, "Enterobacteriaceae," in Manual of Clinical Microbiology, Balows et al.(eds), American Society for Microbiology, [1991], p. 365). Surveillance reports from England, Wales and Ireland for 1986 indicate that E. coli was responsible for 5,473 cases of bacteremia (including blood, bone marrow, spleen and heart specimens); of these, 568 were fatal. For spinal fluid specimens, there were 58 cases, with 10 fatalities (J. J. Farmer and M. T. Kelly, "Enterobacteriaceae," in Manual of Clinical Microbiology, Balows et al.(eds), American Society for Microbiology, [1991], p. 366). There are no similar data for United States, as these are not reportable diseases in this country.
Studies in various countries have identified certain serotypes (based on both the O and H antigens) that are associated with the four major groups of E. coli recognized as enteric pathogens. Table 5 lists common serotypes included within these groups. The first group includes the classical enteropathogenic serotypes ("EPEC"); the next group includes those that produce heat-labile or heat-stable enterotoxins ("ETEC"); the third group includes the enteroinvasive strains ("EIEC") that mimic Shigella strains in their ability to invade and multiply within intestinal epithelial cells; and the fourth group includes strains and serotypes that cause hemorrhagic colitis or produce Shiga-like toxins (or verotoxins) ("VTEC" or "EHEC" [enterohemorrhagic E. coli]).
TABLE 5 ______________________________________ Pathogenic E. coli Serotypes Group Associated Serotypes ______________________________________ Entero- O6:H16; O8:NM; O8:H9; O11:H27; O15:H11; O20:NM; toxigenic O25:NM; O25:H42; O27:H7; O27:H20; O63:H12; O78:H11; (ETEC) O78:H12; O85:H7; O114:H21; O115:H21; O126:H9; O128ac:H7; O128ac:H12; O128ac:H21; O148:H28; O149:H4; O159:H4; O159:H20; O166:H27; and O167:H5 Entero- O26:NM; O26:H11; O55:NM; O55:H6; O86:NM; O86:H2; pathogenic O86:H34; O111ab:NM; O111ab:H2; O111ab:H12; (EPEC) O111ab:H21; O114:H2; O119:H6; O125ac:H21; O127:NM; O127:H6; O127:H9; O127:H21; O128ab:H2; O142:H6; and O158:H23 Entero- O28ac:NM; O29:NM; O112ac:NM; O115:NM; O124:NM; invasive O124:H7; O124:H30; O135:NM; O136:NM; O143:NM; (EIEC) O144:NM; O152:NM; O164:NM; and O167:NM Verotoxin- O1:NM; O2:H5; O2:H7; O4:NM; O4:H10; O5:NM; O5:H16; Producing O6:H1; O18:NM; O18:H7; O25:NM; O26:NM; O26:H11; (VTEC) O26:H32; O38:H21; O39:H4; O45:H2; O50:H7; O55:H7; O55:H10; O82:H8; O84:H2; O91:NM; O91:H21; O103:H2; O111:NM; O111:H8; O111:H30; O111:H34; O113:H7; O113:H21; O114:H48; O115:H10; O117:H4; O118:H12; O118:H30; O121:NM; O121:H19; O125:NM; O125:H8; O126:NM; O126:H8; O128:NM; O128:H2; O128:H8; O128:H12; O128:H25; O145:NM; O125:H25; O146:H21; O153:H25; O157:NM; O157:H7; O163:H19; O165:NM; O165:19; and O165:H25 ______________________________________
A. Verotoxin Producing Strains of E. coli
Although all of these disease-associated serotypes cause potentially life-threatening disease, E. coli O157:H7 and other verotoxin-producing strains have recently gained widespread public attention in the United States due to their recently recognized association with two serious extraintestinal diseases, hemolytic uremic syndrome ("HUS") and thrombotic thrombocytopenic purpura ("TTP"). Worldwide, E. coli O157:H7 and other verotoxin-producing E. coli (VTEC) are an increasingly important human health problem. First identified as a cause of human illness in early 1982 following two outbreaks of food-related hemorrhagic colitis in Oregon and Michigan (M. A. Karmali, "Infection by Verocytotoxin-Producing Escherichia coli," Clin. Microbiol. Rev., 2:15-38 [1989]; and L. W. Riley, et al. "Hemorrhagic colitis associated with a rare Escherichia coli serotype," New Eng. J. Med., 308: 681-685 [1983]), the reported incidence of VTEC-associated disease has risen steadily, with outbreaks occurring in the U.S., Canada, and Europe. In one nursing home outbreak in Canada, 55 elderly residents and 18 staff members were involved, and 17 residents (aged 78 to 99 years) died due to complications of hemorrhagic colitis (C. Krishnan et al., "Laboratory investigation of outbreak of hemorrhagic colitis caused by Escherichia coli 0157:H7," J. Clin. Microbiol., 25:1043-1047 [1987]). This outbreak was notable for its relatively high fatality rate (approximately 31%).
With increased surveillance, E. coli O157:117 has been recognized in other areas of the world including Mexico, China, Argentina, Belgium, and Thailand (N. V. Padhye and M. P. Doyle, "Escherichia coli 0157:H7: Epidemiology, pathogenesis and methods for detection in food," J. Food. Prot., 55: 555-565 [1992]; D. Pierard et al., "Results of screening for verocytotoxin-producing Escherichia coli in faeces in Belgium," Eur. J. Clin. Microbiol. Infect. Dis., 9:198-201 [1990]; and P. M. Griffin and R. V. Tauxe, "The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome," Epidemiol. Rev., 13: 60 [1991]).
The disease attracted national attention in the U.S. after a major outbreak in the Pacific Northwest that was associated with consumption of undercooked E. coli O157:H7-contaminated hamburgers. Over 700 hundred people fell ill (more than 170 were hospitalized) and four young children died (P. I. Tarr, "Review of 1993 Escherichia coli 0157:H7 outbreak: Western United States," Dairy Food & Environ. Sanitation 14:372-373 [1994]; and P. Recer, "Experts call for irradiation of meat to protect against food-borne bacteria," Associated Press, Jul. 12, 1994 [1994]). Several outbreaks since then have underscored the potential severity and multiple mechanisms for transmission of VTEC-associated diseases (M. Bielaszewska et al., "Verotoxigenic (enterohaemorrhagic) Escherichia coli in infants and toddlers in Czechoslovakia," Infection 18: 352-356 [1990]; A. Caprioli et al., "Hemolytic-uremic syndrome and Vero cytotoxin-producing Escherichia coli infection in Italy, " J. Infect. Dis., 166: 184-158 [1992]; A. Caprioli et al., "Community-wide Outbreak of Hemolytic-Uremic Syndrome Associated with Non-O157 Verocytotoxin-Producing Escherichia coli," J. Infect. Dis., 169: 208-211 [1994]; N. Cimolai, "Low frequency of high level Shiga-like toxin production in enteropathogenic Escherichia coli serogroups," Eur. J. Pediatr., 151: 147 [1992]; J. G. Wells, "Laboratory investigation of hemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype," J. Clin. Microbiol., 18:512-520 [1983]; and R. Voelker, "Panel calls E. coli screening inadequate," Escherichia coli O157:H7--Panel sponsored by the American Gastroenterological Association Foundation in July 1994, Medical News & Perspectives, J. Amer. Med. Assoc., 272: 501 [1994]). One 1990 outbreak of interest to those responsible for water quality was associated with water-borne transmission due to freeze-fracture of municipal water pipes. This outbreak required two months to control, was quite large, and notable for its severity, with two HUS cases and four deaths (reviewed by MA. Neill, "E. coli 0157:H7 time capsule: What do we know and when did we know it?," Dairy Food & Environ. Sanitation 14:374-377 [1994]).
While O157:H7 is currently the predominant E. coli serotype associated with illness in North America, other serotypes (as shown in Table 1, and in particular O26:H11, O113:H21, O91:H21 and O111:NM) also produce verotoxins which appear to be important in the pathogenesis of gastrointestinal manifestations and the hemolytic uremic syndrome (P. M. Griffin and R. V. Tauxe, "The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome," Epidemiol. Rev., 13: 60 [1990]; M. M. Levine et al., "Antibodies to Shiga holotoxin and to two synthetic peptides of the B subunit in sera of patients with Shigella dysenteriae 1 dysentery," J. Clin. Microbiol., 30: 1636-1641 [1992]; J. R. Molenda et al.,"Escherichia coli (including 0157:H7): An environmental health perspective," Dairy Food & Environ. Sanitation 14:742-747 [1994]; and C. R. Dorn et al., "Properties of Vero cytotoxin producing Escherichia coli of human and animal origin belonging to serotypes other than O157:H7," Epidemiol. Infect., 103: 83-95 [1989]). Since organisms with these serotypes have been shown to cause illness in humans they may assume greater public health importance over time (P. M. Griffin and R. V. Tauxe, "The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome," Epidemiol. Rev., 13: 60 [1990]).
Clinicians usually observe cases of hemolytic uremic syndrome ("HUS") clustered in a geographic region. However, small outbreaks are likely to be missed because many laboratories do not routinely screen stool specimens for E. coli O157:H7. Many cases related to non-commercial food preparation also probably go unrecognized. Nonetheless, E. coli O157:H7 is responsible for a large number of cases, as more than 20,000 cases of E. coli O157:H7 infection are reported annually in the U.S., with 400-500 deaths from HUS. However, these estimates were compiled when only 11 states mandated reporting of E. coli O157:H7. Twenty-nine states havc recently made E. coli O157:H7 infection a reportable disease (R. Voelker, "Panel calls E. coli screening inadequate; Escherichia coli O157:H7; panel sponsored by the American Gastroenterological Association Foundation in July 1994, Medical News & Perspectives," J. Amer. Med. Assoc., 272: 501 [1994]). Indeed, the Centers for Disease Control recently added E. coli O157:H7 to their list of reportable diseases ("Public Health Threats," Science 267:1427 [1995]), and is now recommending that all diarrheal specimens be examined for its presence.
B. Nature of Verotoxin-Induced Disease
Risk factors for HUS progression following infection with E. coli O157:H7 include age (very young or elderly), bloody diarrhea, leukocytosis, fever, large amounts of ingested pathogen, previous gastrectomy, and the use of antimicrobial agents (in particular, trimethoprim-sulfamethoxazole)(A. A. Harris et al., "Results of a screening method used in a 12 month stool survey for Escherichia coli O157:H7," J. Infect. Dis., 152: 775-777 [1985]; and M. A. Karmali, "Infection by Verocytotoxin-producing Escherichia coli," Clin. Microbiol. Rev., 2: 15-38 [1989]).
As indicated above, E. coli O157:H7 is associated with significant morbidity and mortality. The spectrum of illness associated with E. coli O157:H7 infection includes asymptomatic infection, mild uncomplicated diarrhea, hemorrhagic colitis, HUS, and TTP". Hemorrhagic colitis (or "ischemic colitis") is a distinct clinical syndrome characterized by sudden onset of abdominal cramps--likened to the pain associated with labor or appendicitis--followed within 24 hours by watery diarrhea. One to two days later, the diarrhea turns grossly bloody in approximately 90% of patients and has been described as "all blood and no stool" (C. H. Pai et al., "Sporadic cases of hemorrhagic colitis associated with Escherichia coli O157:H7," Ann. Intern. Med., 101: 738-742 [1984]; and R. S. Remis et al., "Sporadic cases of hemorrhagic colitis associated with Escherichia coli O157:H7," Ann. Intern. Med., 101: 738-742 [1984]). Vomiting may occur, but there is little or no fever. The time from ingestion to first loose stool ranges from 3-9 days (with a mean of 4 days) L. W. Riley et al., "Hemorrhagic colitis associated with a rare Escherichia coli serotype," New Eng. J. Med., 308: 681-685 [1983]; and D. Pudden et al., "Hemorrhagic colitis in a nursing home," Ontario Can. Dis. Weekly Rpt., 11: 169-170 [1985]), and the duration of illness ranges generally from 2-9 days (with a mean of 4 days).
HUS is a life-threatening blood disorder that appears within 3-7 days following onset of diarrhea in 10-15% of patients. Those younger than 10 years and the elderly are at particular risk. Symptoms include renal glomerular damage, hemolytic anemia (rupturing of erythrocytes as they pass through damaged renal glomeruli), thrombocytopenia and acute kidney failure. Approximately 15% of patients with HUS die or suffer chronic renal failure. Indeed, HUS is a leading cause of renal failure in childhood (reviewed by M. A. Karmali, "Infection by Verocytotoxin-producing Escherichia coli," Clin. Microbiol. Rev., 2: 15-38 [1989]). Currently, blood transfusion and dialysis are the only therapies for HUS.
TTP shares similar histopathologic findings with HUS, but usually results in multiorgan microvascular thrombosis. Neurological signs and fever are more prominent in TTP, compared with HUS. Generally occurring in adults, TTP is characterized by microangiopathic hemolytic anemia, profound thrombocytopenia, fluctuating neurologic signs, fever and mild azotemia (H. C. Kwaan, "Clinicopathological features of thrombotic thrombocytopenic purpura," Semin. Hematol., 24: 71-81 [1987]; and S. J. Machin, "Clinical annotation: Thrombotic thrombocytopenic purpura," Br. J. Hematol., 56: 191-197 [1984]). Patients often die from microthrombi in the brain. In one review of 271 cases, a rapidly progressive course was noted, with 75% of patients dying within 90 days (E. L. Amorosi and J. E. Ultmann, "Thrombotic thrombocytopenic purpura: Report of 16 cases and review of the literature," Med., 45:139-159 (1966).
Other diseases associated with E. coli O157:H7 infection include hemorrhagic cystitis and balantitis (W. R. Grandsen et al., "Hemorrhagic cystitis and balantitis associated with verotoxin-producing Escherichia coli O157:H7," Lancet ii: 150 [1985]), convulsions, sepsis with other organisms and anemia (P. C. Rowe et al., "Hemolytic anemia after childhood Escherichia coli O157:H7 infection: Are females at increased risk?" Epidemiol. Infect., 106: 523-530 [1991]).
While the pathogenic mechanism of E. coli O157:H7 infection is incompletely understood, it is believed that ingested organisms adhere to and colonize the intestinal mucosa, where toxins are released which cause endothelial cell damage and bloody diarrhea. It is also postulated that hemorrhagic colitis progresses to HUS when verotoxins enter the bloodstream, damaging the endothelial cells of the microvasculature and triggering a cascade of events resulting in thrombus deposition in small vessels. These microthrombi occlude the microcapillaries of the kidneys (particularly in the glomeruli) and other organs, resulting in their failure (J. J. Byrnes and J. L. Moake, "TTP and HUS syndrome: Evolving concepts of pathogenesis and therapy," Clin. Hematol., 15: 413-442 [1986]; and T. G. Cleary, "Cytotoxin-producing Escherichia coli and the hemolytic uremic syndrome," Pediatr. Clin. North Am., 35: 485-501 [1988]). Verotoxins entering the bloodstream may also result in direct kidney cytotoxicity.
The role of verotoxins in the pathogenesis of E. coli O157:H7 infections has been further studied in animal models. Infection or toxin challenge of laboratory animals do not produce all the pathologies and symptoms of hemorrhagic colitis, HUS, and TTP which occur in humans. Glomerular damage is noticeably absent. Nonetheless, experiments using animal models implicate verotoxins as the direct cause of hemorrhagic colitis, microvascular damage leading to the failure of kidneys and other organs and CNS neuropathies.
In terms of treatment and prognosis, by the time symptoms are serious enough to attract medical attention, it is likely that verotoxins are already entering the systemic circulation or will do so shortly thereafter. Although antimicrobials may help to prevent pathology resulting from the action of toxin on the bowel lumen. However, by the time symptoms of HUS have developed, the patient has ceased shedding organisms. Thus, antimicrobial treatment during HUS disease is of less value, and often contraindicated, due to the increased risk of complications associated with administration of antimicrobials to patients susceptible to development of HUS.
III. Salmonella as a Pathogenic Organism
Worldwide, Salmonella have been isolated from humans and almost all other animal species. However, some serotypes are essentially species specific. For example, the only known natural reservoir of S. typhi is the human population. The same is essentially true for S. paratyphi types A, B, and C. Although these Salmonella are the most widely known, other of the various serotypes of Salmonella are well-documented causes of enteric and systemic disease. Many of these serotypes are associated with various wild, domestic and feral animals. For example, Salmonella infection in poultry is a serious source of food spoilage, and causes food poisoning in as many as four million people per year in the United States (Black, at p. 758).
Salmonella are associated with a wide range of infection and disease, ranging from mild, self-limiting gastroenteritis to life-threatening typhoid fever. The most common form of Salmonella disease is self-limiting gastroenteritis, with fever that lasts less than two days, and diarrhea that lasts less then one week. Typhoid, an enteric fever that is among the best-studied, is characterized by fever, headache, diarrhea, and abdominal pain. Typhoid is also sometimes associated with respiratory, hepatic, splenic, and/or neurologic damage. Other diseases associated with Salmonella include bacteremia,, meningitis, respiratory disease, cardiac disease, osteomyelitis, and local infections. Salmonella infections are particularly dangerous in patients such as the very young, the very old or immunocompromised individuals (e.g., AIDS patients).
Of the numerous Salmonella serotypes, S. typhimurium is most frequently isolated serotype in the United States (See e.g., L. D. Gray, "Escherichia, Salmonella, Shigella, and Yersinia," in P. Murray et al. (eds), Manual of Clinical Microbiology, 6th edition, ASM Press, Washington, D.C., pp. 450-456 [1995]). The nomenclature and classification of the Salmonella have changed many times over the years, and remain unsettled. For example, members of the genus Salmonella and the former genus Arizona are so closely related that they are presently considered to be one species (Salmonella). There is general consensus that there are seven distinct subgroups of Salmonella, with each group exhibiting its own phenotypic characteristics, and historical nomenclature. Most clinical isolates of Salmonella belong in group I. The members of these subgroups are serotyped according to somatic (O), surface (Vi), phase 1 flagellar and phase 2 flagellar (H) antigens. For example Salmonella subgroup 1 (S. choleraesuis) serotype 1,4,5,12:i:1,2, may be referred to as "Salmonella subgroup 1, serotype typhimurium, " or "Salmonella, serotype typhimurium," or "S. typhimurium." The last, simple version has been in common use for many years. Indeed, the CDC has indicated its acceptance of this type of nomenclature, as being widely accepted, practical, and clinically informative (See, Gray at p. 453).
IV. Microbiological Analysis of Water and Food
As discussed above, water and food present significant risks to the public in terms of transmission of infectious disease. However, methods to directly detect and identify pathogens in food and water has proven to be problematic. Problems associated with recovery of pathogens from water and food led to the development of methods to detect and enumerate "indicators" of fecal contamination. These organisms serve to indicate whether a given water supply or food source is contaminated with fecal material, without actually testing for the presence of pathogens. This contamination is viewed as predictive of the potential presence of enteric pathogens (i.e., without the presence of fecal material, the chances of these pathogens being present is usually remote). However, a number of issues remain to be resolved, not the least of which is the significance of the presence of indicator organisms in water supplies.
Historically, "coliforms" have served as the indicator bacteria for fecal contamination in United States water supplies and food production. However, term "coliform" encompasses four genera (Escherichia, Citrobacter, Enterobacter, and Klebsiella); many of these species are commonly found in the environment in the absence of fecal contamination. Although all of these genera may be recovered from domestic sewage in large numbers, only E. coli is consistently and exclusively found in feces (see e.g., A. P. Dufour, "E. coli: the fecal coliform, in A. W. Hoadley and B. J. Dutka, Bacterial Indicators/Health Hazards Associated with Water, [ASTM, Philadelphia, 1976], p. 48). Thus, coliform detections methods are not specific for the determination of whether a water supply or food has been contaminated with fecal matter. Nonetheless, regulations based on detection and enumeration of "total coliforms" have been in effect in the United States since 1914 (i.e., the Treasury Department Standards of 1914; subsequent standards have been promulgated by the U.S. Public Health Service, and presently, by the U.S. Environmental Protection Agency [EPA]).
Recognition of the fact that most of the organisms included in the designation "total coliforms" are not of fecal origin, led to the development of tests to detect "fecal coliforms," for a subgroup of thermotolerant organisms included within the total coliforms. However, this designation is also not specific, as it includes E. coli, as well as various Klebsiella strains. Despite the fact that although there are substantial extra-fecal sources of Klebsiella, and this organism is infrequently found in human feces, the use of the "fecal coliform" designation and tests to identify these organisms remain routine (reviewed by V. J. Cabelli, "Health Effects Criteria for Marine Recreational Waters," EPA-600/1-80-031, [August, 1983], pp.1-12).
Of potential concern in the monitoring of water and food is the observation that E. coli 0157:H7 has an optimum growth temperature that is in the range of nonfecal coliforms (e.g., Klebsiella pneumoniae and Enterobacter aerogenes), rather than the fecal coliforms (E. V. Raghubeer and J. R. Matches, "Temperature range for growth of Escherichia coli serotype 0157:H7 and selected coliforms in E. coli medium," J. Clin. Microbiol., 28:803-805 [1990]; and M. P. Doyle and J. L. Schoeni, "Survival and growth characteristics of Escherichia coli associated with hemorrhagic colitis," Appl. Environ. Microbiol., 48:855-856 [1984]). Thus, routine screening for fecal coliforms by standard procedures utilizing incubation at 44.5.degree. C. would be likely to exclude E. coli 0157:H7.
Furthermore, the correlation between coliform densities in water and the incidence of waterborne disease originally postulated by Kehr and Butterfield in 1943 (R. W. Kehr and C. T. Butterfield, "Notes on the relationship between coliforms and enteric pathogens," Public Health Repts. 58:589-596 [1943]) have not been supported by experimental tests (Batik et al., "Routine monitoring and waterborne disease outbreaks," J. Environ. Health 45:227-230 [1984]). Quite simply, there has been no direct evidence presented that the level of coliform contamination correlates well with waterborne disease outbreaks (see Pipes, p. 434-435). Nonetheless, due to the lack of better methods, the detection of coliforms as indicator bacteria continues into the present.
Coliform detection may be accomplished by various methods, including multiple tube fermentation (i.e., most probable number or "MPN" determinations), membrane filtration, the "presence-absence" test, and various rapid enzyme (e.g., the MUG test) and immunoassay methods. Important considerations with these methods include the large time, equipment and personnel commitment necessary to conduct and interpret these tests.
Most Probable Number (MPN). The MPN method is a labor, time and supply intensive method, which involves three distinct stages of specimen processing (the presumptive (with lauryl tryptose broth), completed (with brilliant green lactose bile broth) and confirmed tests (with LES Endo or EMB). The MPN method requires 3-4 days in order to produce confirmatory results, and statistical analysis to quantitate the organisms present.
This procedure has been developed to separate organisms within the coliform group into "total" and "fecal" coliforms. Prior enrichment of organisms in a presumptive test medium is required for optimum recovery of fecal coliforms. These methods are used as confirmatory tests conducted with various selective media and elevated incubation temperatures (e.g., 44.5.degree. C.). Thus, there is also a significant time and labor commitment associated with these methods.
Membrane Filtration. In membrane filtration, a known volume of water sample is passed through a membrane filter which is then placed on growth media (e.g., M-Endo or LES-Endo), and incubated overnight. All colonies with characteristics common to coliforms are considered to be members of the coliform group. An advantage of membrane filtration is that preliminary results are usually available in 24 hours. However, verification of colony identification is recommended, usually requiring additional days in order to conduct the needed biochemical tests.
Membrane Filtration Method Modifications. A seven hour fecal coliform test similar to the membrane filtration process has also been described. In this technique, the water sample is filtered and the filter placed on M-7 FC agar and incubated at 41.5.degree. C. (American Public Health Association-American Water Works Association-Water Pollution Control Federation, Standard Methods for the Examination of Water and Wastewater, 16th ed., [APHA, Washington, D.C.], 1985; hereinafter, "Standard Methods"). Yellow colonies representing fecal coliforms are enumerated after seven hours of incubation. However, different growth rates of colonies necessitate a compromise between sensitivity of detection and enumeration. That is to say, because different organisms grow at different rates, some organisms will not have had sufficient time to produce visible colonies on the medium by the time enumeration is conducted. However, the value of this test is perhaps questionable, in view of its deletion from the most recent edition of Standard Methods.
Another method developed by Reasoner, in conjunction with Geldreich (D. J. Reasoner and E. E. Geldreich, "Rapid detection of water-borne fecal coliforms by .sup.14 CO.sub.2 release," in A. N. Sharpe and D. S. Clark, (eds.) Mechanizing Microbiology, [Charles C. Thomas Publishers, 1978], pp. 120-139) involves concentration of bacteria on a membrane filter which is then placed in M-FC broth which contains radiolabelled .sup.14 C-mannitol. The tubes are incubated for 2 hours at 35.degree. C., followed by 2.5 hours at 44.50. Release of .sup.14 CO.sub.2 due to microbial metabolism is then assayed by liquid scintillation spectrometry. Major problems with these methods involve the use of radioactivity and the attendant disposal and handling concerns, as well as the need for specialized and expensive instruments.
An alternate radioactive test was developed by Dange el al. (V. Dange et al., "One hour portable test for drinking waters," Water Res., 22:133-137 [1988]). This method is based on the correlation of .sup.32 P uptake by organisms present in a water sample incubated in a synthetic medium. Thus, these methods require highly trained laboratory personnel and are not suitable for use in many labs.
Presence-Absence Test. The presence-absence test to detect the presence of coliforms involves the inoculation of broth with 100 ml samples of water, followed by incubation at 25.degree. C. for 24-48 hours. If acid and gas is produced in the medium, the test is positive for the presence of coliforms (see e.g., Standard Methods, at p. 882-884). No enumeration of organisms is attempted, nor are any identification methods utilized. Thus, the information garnered from this method is very limited.
Fluorometric and Enzymatic Tests. Detection methods for coliforms with fluorometric tests and numerous variations on the basic technology have also been developed. Other substrate-based methods include the use of such compounds as ortho-nitrophenyl-.beta.-D-galactopyranoside (ONPG), 5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronide (X-GLUC) (see e.g., E.W. Frampton et al., "Evaluation of the .beta.-glucuronidase substrate 5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronide (X-GLUC) in a 24-hour direct plating method for Escherichia coli," J. Food Protect., 51:402-404 [May 1988]; and L. Restaino et al., "Use of the chromogenic substrate 5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronide (X-GLUC) for enumerating Escherichia coil in 24 H from ground beef," J. Food Protect., 53:508-510 [1990]);, and 4-methylumbelliferyl-.beta.-D-glucuronide (MUG). These methods utilize fluorogenic or chromogenic substrates to detect coliform metabolism, as opposed to direct detection and enumeration of organisms. Thus, the only data available from these test methods relate to the presence or absence of organisms which possess the necessary enzymatic machinery to produce the detectable color compounds from a given substrate.
The MUG test is also problematic in that many clinically important E. coli strains are negative (see e.g., E. W. Frampton and L. Restaino, "Methods for Escherichia coli identification in food, water and clinical samples based on beta-glucuronidase detection," J. Appl. Bacteriol., 74:223-233 [1993]). Indeed, there is a significant proportion of .beta.-glucuronidase-negative E. coli (see e.g., G. W. Chang et al., "Proportion of .beta.-D-glucuronidase-negative Escherichia coli in human fecal samples," Appl. Environ. Microbiol., 55:335-339 [1989]). Furthermore, species within other genera such as Staphylococcus, Streptococcus, Clostridium, and the anaerobic corynebacteria also produce .beta.-glucuronidase (Frampton and Restaino, p. 223). Thus, not only is the test not highly sensitive, it is not specific. These reports raise serious questions regarding the reliability of these testing methods.
Bacteriophages. In addition to culture and enzymatic detection methods, bacteriophages have also been used with some limited success as indicators of fecal contamination (R. S. Wensel et al., "Evaluation of coliphage detection as a rapid indicator of water quality," Appl. Environ. Microbiol., 43:430-434 [1982]; Y. Kott et al., "Bacteriophages as bacterial viral pollution indicators," Water Res., 8:165-171 [1982]; and A. H. Havelaar et al., "Factors effecting the enumeration of coliphages in sewage and sewage-polluted waters," Antonie van Leewenhoek 49:387-397 [1983]).
However, the detection limits provided by these methods are no better than those obtained with standard methods for water quality determinations based on coliform analysis. Thus, these methods do not provide a significant advantage over the traditional methods of water analysis. Likewise, the presence of coliforms in a particular water or food sample does not necessarily correlate well with the incidence of disease. Even the enumeration of "fecal coliforms" is less than optimal, as some organisms such as Klebsiella are capable of producing positive test results. Such observations led to the development of alternative indicator organisms, including tests specific for E. coli, fecal streptococci (e.g., enterococci), Klebsiella, Clostridium perfringens, Pseudomonas aeriginosa, Bifidobacterium, Bacteroides, Aeromonas hydrophila, V. parahaemolyticus, and C. albicans, as well as other organisms commonly excreted in large numbers by healthy mammals. What remains to be developed is a method for the detection and enumeration of pathogens commonly associated with water and food-borne diarrheal illness.
V. Diagnosis of Enteric Disease/Infection
The diagnosis of infectious disease has traditionally relied upon microbiological culture methods to identify the causative organism and determine the appropriate antimicrobial treatment. This has remained so despite recent advances in molecular and immunological diagnostics. While the development of rapid and automated methods has served to increase the efficiency of microbiological analysis, traditional methods remain critical for definitive diagnosis of infections (Baron & Finegold, p. 253). As discussed above, isolation and identification of pathogens in environmental samples, food, and water present additional considerations. For example, the methods available for detecting E. coli 0157:H7 in food are extremely time-consuming, or are not highly specific (N. V. Padhye and M. P. Doyle, "Rapid procedure for detecting enterohemorrhagic Escherichia coli 0157:H7 in food," Appl. Environ. Microbiol., 57:2693-2698 [1991]). Nonetheless, clinical, as well as food, water, and environmental samples must be analyzed for the presence of pathogens.
After proper specimen collection and transport, the laboratory professional must determine which of a multitude of culture media are most appropriate to use with the culture at hand. It is important to consider the type of specimen (e.g, urine, blood, sputum, etc.), and the most commonly isolated organisms associated with disease or infection at the site of specimen collection. The time and cost necessary to achieve a final diagnosis also must be borne in mind. For gastrointestinal samples (e.g, fecal samples, rectal swabs, colonoscopy samples, etc.) the presence of a large complement of commensal organisms in the gastrointestinal tract necessitates the use of media and procedures to optimize growth of pathogenic organisms, while differentiating them from the normal flora. Numerous organisms are included among the normal flora of healthy individuals, including S. epidermidis, S. aureus, viridans streptococci, enterococci, S. pyogenes and other streptococci, peptostreptococci, lactobacilli, corynebacteria, mycobacteria, clostridia, actinomycetes, various members of the Enterobacteriaceae, P. aeruginosa, Alcaligenes faecalis, Flavobacterium spp., Bacteroides spp., Fusobacterium spp., Eubacterium spp., Propionibacterium spp., Bifidobacterium spp., yeasts, filamentous fungi, and various protozoans (e.g., Entamoeba coli, Endolimax nana, Iodamoeba butschlii, Trichomonas hominis, and Chilomaslix mesnili) (see e.g., H. D. Isenberg and R. F. D'Amato, "Indigenous and Pathogenic Microorganisms of Humans," in P. Murray et al. (eds), Manual of Clinical Microbiology, 6th edition, ASM Press, Washington, D.C., [1995]), p.9). Enrichment techniques are often required for successful isolation of Salmonella, Shigella and Vibrio. These enrichment media work on the principle that normal fecal flora are maintained in a prolonged lag phase by inhibitory compounds present in the growth media, while the organism enriched for (e.g., Salmonella and Shigella) are far less inhibited, enter the log phase of growth and are more readily recovered (See e.g., E. W. Koneman et al., Color Atlas and Textbook of Diagnostic Microbiology, 4th ed., J. B. Lippincott Co., Philadelphia [1992], p. 119).
Traditionally, it has been recommended that laboratories routinely examine specimens for the presence of Salmonella, Shigella, Campylobacter, Aeromonas, and Plesiomonas, as well as predominating numbers of S. aureus, yeasts, Pseudomonas spp., Yersinia spp., and some Vibrio spp. from cases of gastrointestinal disease (,See e.g., A. Grasmick, "Processing and Interpretation of Bacterial Fecal Cultures," in "Section I. Aerobic Bacteriology," in H. Isenberg (ed.), Clinical Microbiology Procedures Handbook, American Society for Microbiology, Washington, D.C., [1994], p. 1.10.5; and Baron and Finegold, at 61, 247-251). Each laboratory must assess the patient history, the patient population associated with the laboratory or hospital, the geographic location of the facility, and hospital size in order to choose the selective and differential media that will be routinely used to culture fecal samples. Routine cultures for isolation of enteric organisms from clinical specimens usually include a blood agar plate (for aerobic incubation), at least one differential medium (e.g., MacConkey and EMB agars), at least one moderately selective medium (e.g., Hektoen-enteric (HE agar), Xylose-lysine ]deoxycholate (XLD agar), Salmonella-Shigella (SS agar), desoxycholate, desoxycholate-citrate, and desoxycholate-lactose agars), and at least one enrichment broth (e.g., tetrathionate broth, selenite F broth, selenite cystine broth, Hajna broth, and Gram-negative broth (GN)). Media for selective isolation and enrichment of Campylobacter (e.g., Skirrow's, Blaser's, Butzler's, or Preston's formulae, and Campy-THIO agars) and special incubation conditions (i.e., a microaerophilic atmosphere and 42.degree. C.) are also included, as are media for Aeromonas (a blood agar plate with 10 .mu.g/ml ampicillin), Plesiomonas (inositol-brilliant-green bile-salts agar), and Yersinia (cefsulodin-irgasan-novobiocin agar [CIN]), and Vibrio (thiosulfate citrate bile salts agar [TCBS], and peptone broth for enrichment). Additional plates sometimes include media selective for C. difficile (cycloserine-cefoxitin-fructose-egg yolk agar [CCFA]). In the investigation of an outbreak, at least one highly selective medium (e.g., brilliant green, and bismuth sulfite agars), as well as any other media and incubation conditions indicated by the type of disease (e.g., special anaerobic media and anaerobic incubation, media for isolation of mycobacteria, or media for isolation of Helicobacter pylori) are added to the battery of media inoculated. Thus, for each sample, numerous plated and broth media are inoculated for primary isolation of enteric organisms. Many of the same media are routinely used for isolation and identification of pathogenic organisms from food, dairy and water samples, especially in outbreak situations.
Selective Media for E. coli and Salmonella Species
In 1993, the CDC recommended that all laboratories routinely culture for E. coli 0157:H7 from all patients presenting with diarrhea (See, Gray, at p. 452). Fecal samples should be cultured within 7 days of the onset of intestinal illness in adults and within 30 days of onset of intestinal illness in children (See, Gray at p. 452). More emphatically, fecal samples should be collected from any patient who reports having bloody diarrhea, and tested for E. coli 0157:H7.
MacConkey-sorbitol agar (SMAC) is the most commonly used medium for isolation of E. coli 0157:H7 and provides a preliminary characterization of E. coli isolates suspected of being E. coli 0157:H7 (S. B. March and S. Ratnam, "Sorbitol-MacConkey medium for detection of Escherichia coli 0157:H7 associated with hemorrhagic colitis," J. Clin. Microbiol., 23:869-872 [1986]). E. coli colonies that do not ferment sorbitol (i.e., non-colored colonies) may be further characterized by serotyping and/or testing for verotoxin production. Nearly all E. coli 0157:H7 isolates do not ferment sorbitol, whereas most non-pathogenic E. coli isolates ferment this substrate.
Unfortunately, SMAC has a very major drawback in that it is not highly specific for detecting E. coli 0157:H7. One problem is that other sorbitol-negative E. coli will also be detected as false positives on this medium. Indeed, as many as 20% of clinical isolates of E. coli may be sorbitol-negative (NCASM Winter Newsletter, [1994] p. 4). In addition, many other species such as Aeromonas, Budvicia aquatica, Buttiauxella agrestis, Cedecea davisae, Enterobacter agglomerans biogroups 2, 3, 4, and 6, E. laylorae, Erwinia carotovora, Escherichia blattae, E. alkalescens-dispar, E. fergusonii, E. hermanii, E. vulneris, Ewingella americana, Klebsiella pneumoniae ss. rhinoscleromatis, Kluyvera cryocrescens, Lecleria adecarboxylata, Leminorella grimontii, L. richardii, Morganella morganli, Obesumbacterium proteus biogroup 2, Plesiomonas shigelloides, Pragia fontium, Proteus mirabills, P. myxofaciens, P. vulgaris, Providencia alcalifaciens, P. heimbachae, P. rettgeri, P. rustiganni, P. stuartii, Serratia entomophila, S. plymuthica, Shigella boydii, S. flexneri, S. sonnel, Xenorhabdus nematophilus, Yersinia pestis, Y pseudotuberculosis, Y ruckeri, and Yokenella regensburgei also grow as non-colored colonies on SMAC.
Furthermore, some rare isolates of E. coil 01 57:H7 have been reported as being sorbitol-positive (See e.g., P. M. Frantamico et al., "Virulence of an Escherichia coli 0157:H7 sorbitol-positive mutant," Appl. Environ. Microbiol., 59:4245-4252). Thus, these sorbitol-positive isolates would be assumed to be normal fecal flora, and not be considered for further identification and testing upon their isolation on SMAC and it is likely that the patient's disease would be undiagnosed.
An ideal medium would also detect other E. coli serotypes, in addition to E. coli 0157:H7, as other serotypes have been shown to produce verotoxins or other shiga-like toxins, and have also been associated with clinical disease (See Table 5). However, these other serotypes are not differentiated on SMAC because they are sorbitol-positive (see e.g., M. Ritchie et al., "Comparison of a direct fecal shiga-like toxin assay and sorbitol MacConkey agar culture for laboratory diagnosis of enterohemorrhagic Escherichia coli infection," J. Clin. Microbiol., 30:461-464 [1992]).
In addition to their characteristic inability to ferment sorbitol, most E. coli 01 57:H7 isolates do not produce .beta.-glucuronidase, and cannot cleave glucuronidase substrates such as 4-methylumbelliferyl-.beta.-D-glucuronide (MUG). This property also serves as a means to help differentiate many non-pathogenic E. coli isolates from non-0157:H7, as most of the non-pathogenic isolates are capable of cleaving MUG to produce a fluorescent product that is visible under UV light (i.e., these isolates are MUG-positive, while E. coli 0157:H7 is MUG-negative; J. S. Thompson et al., "Rapid biochemical test to identify verocytotoxin-positive strains of Escherichia coli serotype 0157," J. Clin. Microbiol., 28:2165-2168 [1990]). However, the fluorescent product in this test diffuses out of the colonies, obscuring the actual colonial source of the enzyme.
Some researchers have investigated the addition of chromogenic glucuronidase substrates such as 5-bromo-4-chloro-3-indoxyl-.beta.-glucuronide (X-GLUC) to SMAC (A. J. G. Okrend et al., "Use of 5-bromo-4-chloro-3-indoxyl-.beta.-glucuronide in SMAC to aid in the isolation of Escherichia coli 0157:H7 from ground beef," J. Food Protect., 53:941-943 [1990]; and F. Nimoorand and C. Lord, J. Rapid Meth. Automation Microbiol., 3:85-96 [1994]). However, these media only help differentiate .beta.-glucuronidase-positive, sorbitol-negative E. coli isolates. It does not differentiate other glucuronidase-negative, sorbitol-negative E. coli isolates, or any of the other species that grow as non-colored colonies on SMAC discussed previously. Thus, additional work is necessary to differentiate between E. coli 0157:H7 and other species which grow as non-colored colonies on this medium.
In yet another SMAC-based medium for E. coli 0157:H7, rhamnose and cefixime are incorporated in SMAC. This medium has been reported to be somewhat beneficial, as cefixime inhibits Proteus, and rhamnose is fermented by most non-sorbitol fermenting E. coli serogroups other than 0157:H7 (P. A. Chapman et al., "An improved selective medium for the isolation of Escherichia coli 0 157," J. Med. Microbiol., 35:107-110 [1991]). This medium has a disadvantage in that although the false positive rate is lower compared with regular SMAC, numerous colonies are still isolated that are not E. coli 0157:H7. Furthermore, many E. coil 0157:H7 isolates are rhamnose-positive (S. L. Abbott et al., "Escherichia coli 0157:H7 generates a unique biochemical profile on MicroScan conventional gram-negative identification panels," J. Clin. Microbiol., 32:823-824 [1994]) and would not be detected on this medium.
Still another variation incorporates tellurite and cefixime in SMAC (P. M. Zadik et al., "Use of tellurite for the selection of verocytotoxigenic Escherichia coli 0157," J. Med. Microbiol., 39:155-158 [1993]). However, even the tellurite-containing medium had a high rate of false positives ("[t]he suppression of non-0157 E. coli on TC-SMAC uncovered a large number of NSF [non sorbitol fermenting] colonies (206 among the 391 specimens) that required screening by latex agglutination for 0157."
Several non-MacConkey sorbitol-containing media have been developed for isolation and preliminary identification of E. coil 0157:H7. One medium incorporates tryptone, sorbitol, sodium chloride, bile salts, bromcresol purple, and MUG (R. A. Szabo et al., "Method to isolate Escherichia coli 0157:H7 from food," J. Food Protect., 49:768-772 [1986]). Organisms are grown overnight at 44.5.degree. C. on membrane filters placed on top of the agar medium. This method also depends upon the non-utilization of sorbitol to differentiate between E. coil 0157:H7 and other serotypes. On this medium, E. coli 0157:H7 is reported as being sorbitol-negative, MUG-negative, and indole-positive. Non-0157:H7 E. coil are sorbitol-positive, MUG-positive, and appear to be indole-negative, due to utilization of sorbitol, which is preferentially metabolized over tryptophan (in the tryptone). However, upon prolonged incubation, these colonies can appear to be indole-positive. As this method uses incubation at an elevated temperature, it is possible that many isolates of E. coil 0157:H7 would not be detected (E. V. Raghubeer and J. R. Matches, "Temperature range for growth of Escherichia coli serotype 0157:H7 and selected coliforms in E. coil medium," J. Clin. Microbiol., 28:803-805 [1990]; and M. P. Doyle and J. L. Schoeni, "Survival and growth characteristics of Escherichia coli associated with hemorrhagic colitis," Appl. Environ. Microbiol., 48:855-856 [1984]). In addition, some types of membrane filters appear to inhibit some E. coli 0157:H7 isolates.
Another modified sorbitol-containing medium has also been described, which incorporates antiserum directed against H7 (J. J. Farmer and B. R. Davis, "H7 antiserum-sorbitol fermentation medium: A single tube screening medium for detecting Escherichia coli 0157:H7 associated with hemorrhagic colitis," J. Clin. Microbiol., 22:620-625 [1985]). This medium is highly selective for E. coli 0157:H7, as only about 10% of other E. coli strains have the H7 antigen and about 95% of the non-01 57:H7 E. coli strains ferment sorbitol. However, it requires production and ready supply of anti-H7 antiserum, special handling to incorporate the antiserum into the medium, and would likely to be expensive if made commercially available.
Still another approach utilizes enrichment in EC broth with novobiocin for 24 hours (for optimal results), followed by isolation on several media, (e.g., SMAC, phenol red sorbitol agar with MUG, and EMB) (A. J. G. Okrend et al., "A screening method for the isolation of Escherichia coli 0157:H7 from ground beef," J. Food Protect., 53:249-252 [1990]). This method requires a minimum of two days and multiple plates of media, in order to obtain an identification. It is slow, labor-intensive and also suffers from lack of specificity (as described previously for the other methods).
Other methods, such as immunoassays have been developed to screen samples such as food for the presence of E. coli 0157:H7 (see e.g., M. S. Kim and M. P. Doyle, "Dipstick immunoassay to detect enterohemorrhagic Escherichia coli 0157:H7 in retail ground beef," Appl. Environ. Microbiol., 58:1764-1767 [1992]). However, these methods are time-consuming, require the production of monoclonal antibodies and the optimization of such immunoassay systems as ELISA's. In addition, immunological methods are not likely to detect all verotoxins with equal efficiency and may not detect some at all (Gannon et al., "Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction," Appl. Environ. Microbiol., 58:3809-3815 [1992]).
Molecular methods, such as the polymerase chain reaction (PCR) have been used to differentiate between types or variants of verotoxin genes (M. P. Jackson, "Detection of Shiga toxin-producing Shigella dysenteriae type 1 and Escherichia coli using the polymerase chain reaction with incorporation of digoxigenin- 11-dUTP," J. Clin. Microbiol., 29:1910-1914 [1991]; W. M. Johnson et al., "Amplification by the polymerase chain reaction of a specific target sequence in the coding for Escherichia coli verotoxin (VTe) variant," FEMS Microbiol. Lett., 84:227-230 [1991]; and H. Karch and T. Meyers, "Single primer pair for amplifying segments of distinct Shiga-like toxin genes by polymerase chain reaction," J. Clin. Microbiol., 287:2751-2757 [1989]). There are a few reports of development of PCR methods to detect verotoxin gene sequences in samples such as ground beef (see e.g., Gannon el al.). However, these molecular methods require equipment (e.g., thermal cyclers, etc.), and expertise that this not commonly available in most microbiology laboratories.
Some methods, such as cell culture-based verocytotoxin assays have been developed to detect the presence of toxin, rather than the presence of organisms. However, these cell culture methods require the maintenance of cell cultures, a burden that cannot be met by many laboratories, especially food, water, and environmental laboratories. Indeed, cell cultures are often maintained by virology laboratories, rather than microbiology laboratories (i.e.,traditional bacteriology laboratories). Many laboratories do not have the specialized equipment and expertise necessary to maintain cell cultures. Thus, these methods are not readily adaptable to field situations nor other settings where minimal laboratory capabilities are common.
Thus, better methods for isolation and identification of E. coli 0157:H7 are needed. Indeed, after completing their recent survey of methodologies, Nimoorand et al., concluded that "[a] better enrichment medium, as well as improved selective plating and confirmation techniques, are needed to enhance the selective growth of E. coli 0157:H7 and provide lower detection levels," F. Nimoorand and C. Lord, J. Rapid Meth. Automation Microbiol., 3:85-96 [1994]).
Additional media of more general use in isolation and enumeration of E. coli are disclosed in such patents as U.S. Pat. No. 3,870,601 to Warren et al. (herein incorporated by reference), which describes a culture medium for differentiation of Enterobacteriaceae based on a combination of a chromogenic .beta.-galactosidase substrate, along with substrates for decarboxylase, deaminase, and/or urease, a hydrogen sulfide detection system, and/or a carbohydrate fermentation system. Another medium is disclosed in U.S. Pat. No. 4,070,247 to Burt (herein incorporated by reference), which reduces the incidence of false negative lactose fermentation results, and permitting differentiation between lac inducible and lac constitutive bacteria due to incorporation of isopropyl-.beta.-D-thiogalactopyranoside in the medium. Another medium is disclosed in U.S. Pat. No. 5,210,022 to Roth et al., which utilizes chromogenic .beta.-galactosidase substrate and chromogenic .beta.-glucuronidase substrates to differentiate between coliforms (species other than E. coli) and E. coli. Yet another medium is disclosed in PCT Appln. No. PCT/FR93/00988 (WO 94/08043) to Rambach (herein incorporated by reference), which utilizes a chromogenic glucuronidase substrate to isolate and enumerate E. coli.
Various media and methods have also been developed, with varying degrees of success, for the isolation and identification of Salmonella species. This is exemplified by the numerous media formulations listed in Section V above, which are commonly used in clinical microbiology laboratories to selectively enrich, isolate and differentiate Salmonella.
In a very recent study (H. Dusch and M. Altwegg, "Evaluation of five new plating media for isolation of Salmonella species," J. Clin. Microbiol., 33:802-804 [1995]), HE (BBL) was compared with Rambach agar (Ra; E. Merck, Darmstadt, Germany), SM-ID medium (SM; bioMerieux), XLT4 (xylose-lysine-Tergitol.RTM.-4 agar; Difco); brilliant green-glycerol-lactose agar (NGBL; not commercially available; D. M. Poisson, "Novobiocin, brilliant green, glycerol lactose agar: A new medium for the isolation of Salmonella strains," Res. Microbiol., 143:211-216 [1992]), and modified semisolid Rappaport Vassiliadis medium (MSRV; Difco). The XLT4 medium used in this study is described in U.S. Pat. No. 5,208,150, to Tate et al. (herein incorporated by reference). Rambach agar used in this study is described in Rambach, "New plate medium for facilitated differentiation of Salmonella spp. from Proteus spp. and other enteric bacteria," Appl. Environ. Microbiol., 56:303-303 [1990]; see also, U.S. Pat. Nos. 5,098,832 and 5,194,374 to Rambach).
These authors found that MSRV was the most sensitive of the media tested, and was very specific for the isolation of non-S. typhi from stool specimens. However, the semisolid nature of the medium was found to be a disadvantage and required careful handling in the laboratory. In addition, MSRV, as well as XLT4 and Ra are not suitable for use in the isolation of S. typhi and S. paratyphi type A (Dusch and Altwegg, at p. 804). Thus, these important organisms would be missed, if these media were used exclusively.
Additional media have been tested for their suitability to detect and provide a preliminary identification of Salmonella species in general, and S. typhi in particular. In one study, a lysine-mannitol-glycerol agar (LMG) was developed for isolation of Salmonella, including S. typhi (J. M. Cox, "Lysine-mannitol-glycerol agar, a medium for the isolation of Salmonella spp., including S. typhi and atypical strains," Appl. Environ. Microbiol., 59:2602-2606 [1993]). This medium combines the characteristics of XLD and mannitol-lysine-crystal violet-brilliant green agar, with the addition of glycerol to aid in the differentiation of Salmonella and Citrobacter. The medium facilitates detection of strains with atypical fermentation patterns, such as the lactose or sucrose-positive Salmonella. However, it was not suitable for the isolation of other atypical strains, such as hydrogen sulfide-negative and lysine decarboxylase-negative strains. Also, there was a high percentage of false positive Citrobacter isolates which were observed on this medium. In addition, enrichment was found to be required for optimal detection of S. typhi.
A further modification of LMG developed for isolation of Salmonella from clinical specimens incorporates sulphamandelate in lysine mannitol glycerol agar [LMGS] (K. R. Stallard and J. M. Cox, "Lysine mannitol glycerol agar (LMG) and LMG with sulfphamandelate for isolation of Salmonella spp. from clinical specimens," Lett. Appl. Microbiol., 19:83-87 [1994]). In a comparison of this medium with LMG, it was found to be superior, in terms of sensitivity of detection and selectivity for Salmonella. Although this medium performed better than LMG, a high percentage of false positive Citrobacter isolates was observed. In addition, this medium was only used in conjunction with enrichment methods. Thus, its suitability as a primary isolation medium without a pre-enrichment step is unknown.
In summary, despite a long-standing recognized need and the efforts of many pepole to develop better enrichment and isolation media for E. coli and Salmonella species in general, and E. coli 0157:H7 in particular, all of the media developed to date have important drawbacks and limitations. Indeed, there remains a critical need for media suitable for use in food, water, veterinary, and clinical testing, which would provide rapid, specific and reliable results (See e.g., N. V. Padhye and M. P. Doyle, "Escherichia coli 0157:H7: Epidemiology, pathogenesis, and methods for detection in food," J. Food Protect., 55:555-565 [1992]).